猪囊尾蚴排泄分泌抗原Ts8B3基因的原核表达、纯化以及免疫反应性分析
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摘要
目的将猪囊尾蚴排泄分泌抗原Ts8B3基因进行原核表达,分析重组蛋白的免疫反应性。方法根据Ts8B3基因序列设计引物,以囊尾蚴RNA反转录的cDNA为模板PCR扩增Ts8B3基因,扩增产物经BamHⅠ/XhoⅠ双酶切后连接至原核表达载体pGEX-4T-1,将重组质粒转化入大肠埃希菌DH5α(E.coli DH5α),PCR、双酶切筛选阳性质粒,测序确认后转化至E.coli BL21,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表达产物。纯化重组蛋白,以囊虫病人血清为一抗,采用Western blot分析其免疫反应性。结果 PCR扩增Ts8B3基因片段为201bp,双酶切和测序显示重组表达质粒构建成功。重组质粒转化BL21后经IPTG诱导4h,以包涵体的形式表达相对分子质量单位(Mr)为33×103的目的蛋白。纯化的重组蛋白能被囊虫病患者血清识别。结论成功构建Ts8B3基因重组质粒,表达的重组蛋白具有免疫反应性,为进一步研究该蛋白在抗体检测中的应用奠定了基础。
Objectives To express Ts8B3,a cysticercus-secreted antigen gene,in a prokaryotic expression system and to analyze the immunoreactivity of a recombinant protein. Methods Primers were designed in accordance with the sequence of the Ts8B3 gene,and fragments were amplified with PCR using cDNA from cysticerci as a template.PCR products were digested with BamHI/XhoI and ligated into the prokaryotic expression vector pGEX-4 T-1.The recombinant plasmids were transformed into E.coli DH5α,and positive plasmids were screened using PCR and double enzyme digestion.After they were verified with sequencing,the plasmids were transformed into E.coli BL21 and their expression was induced with isopropyl-β-D-thiogalactopyranoside(IPTG).Protein expression was detected using sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE).The recombinant protein was purified and the serum from a patient with cysticercosis patient was used as the primary antibody.Western blotting was used to analyze the immunoreactivity of the protein. Results Amplification of the Ts8B3 gene with PCR yielded a fragment of 201 bp.Double enzyme digestion and sequencing indicated that the recombinant plasmid was successfully constructed.The recombinant plasmid was transformed into BL21,and its expression was induced with IPTG for 4 h.The target protein with a relative molecular mass(Mr)of 33×10~3 was expressed as an inclusion body.The purified recombinant protein was recognized by serum from a patient with cysticercosis. Conclusion A recombinant plasmid carrying the Ts8B3 gene was successfully constructed,and the recombinant protein was immunoreactive.This work has laid the foundation for further study of the use of this protein in antibody detection.
Objectives To express Ts8B3,a cysticercus-secreted antigen gene,in a prokaryotic expression system and to analyze the immunoreactivity of a recombinant protein. Methods Primers were designed in accordance with the sequence of the Ts8B3 gene,and fragments were amplified with PCR using cDNA from cysticerci as a template.PCR products were digested with BamHI/XhoI and ligated into the prokaryotic expression vector pGEX-4 T-1.The recombinant plasmids were transformed into E.coli DH5α,and positive plasmids were screened using PCR and double enzyme digestion.After they were verified with sequencing,the plasmids were transformed into E.coli BL21 and their expression was induced with isopropyl-β-D-thiogalactopyranoside(IPTG).Protein expression was detected using sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE).The recombinant protein was purified and the serum from a patient with cysticercosis patient was used as the primary antibody.Western blotting was used to analyze the immunoreactivity of the protein. Results Amplification of the Ts8B3 gene with PCR yielded a fragment of 201 bp.Double enzyme digestion and sequencing indicated that the recombinant plasmid was successfully constructed.The recombinant plasmid was transformed into BL21,and its expression was induced with IPTG for 4 h.The target protein with a relative molecular mass(Mr)of 33×10~3 was expressed as an inclusion body.The purified recombinant protein was recognized by serum from a patient with cysticercosis. Conclusion A recombinant plasmid carrying the Ts8B3 gene was successfully constructed,and the recombinant protein was immunoreactive.This work has laid the foundation for further study of the use of this protein in antibody detection.
引文
[1]蔡娟,汪学龙,沈继龙.猪囊尾蚴病免疫诊断研究与进展[J].热带病与寄生虫学,2004,2(2):123-6.
[2]Espindola NM,Vaz AJ,Pardini AX,et al.Excretory/secretory antigens(ES)from in vitro cultures of Taenia crassiceps cysticerci,and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis[J].Ann Trop Med Parasitol,2002,96(4):361-8.
[3]王中全,崔晶.旋毛虫排泄分泌抗原的研究进展[J].中国预防兽医学报,2003,25(2):155-8.
[4]Ev LV,Maia AA,Pianetti G,et al.Immunological evaluation of a 26-kDa antigen fromTaenia soliumlarvae for specific immunodiagnosis of human neurocysticercosis[J].Parasitol Res,1999,85(2):98-102.
[5]Ito A,Plancarte A,Ma L,et al.Novel antigens for neurocysticercosis:simple method for preparation and evaluation for serodiagnosis[J].Am J Trop Med Hyg,1998,59(2):291-4.
[6]Yang HJ,Chung JY,Yun DH,et al.Immunoblot analysis of a10kDa antigen in cyst fluid of Taenia solium metacestodes[J].Parasite immunol,1998,20(10):483-8.
[7]Ko RC,Ng TF.Evaluation of excretory/secretory products of larval Taenia soliumas diagnostic antigens for porcine and human cysticercosis[J].J Helminthol,1998,72(02):147-54.
[8]Molinari JL,Garcia-Mendoza E,de la Garza Y,et al.Discrimination between active and inactive neurocysticercosis by metacestode excretory/secretory antigens of Taenia soliumin an enzymelinked immunosorbent assay[J].Am J Trop Med Hyg,2002,66(6):777-81.
[9]Del Brutto OH,Wadia NH,Dumas M,et al.Proposal of diagnostic criteria for human cysticercosis and neurocysticercosis[J].J Neurol Sci,1996,142(1):1-6.
[10]杜娟,牟荣,包怀恩,等.雷丸、阿苯达唑和吡喹酮对亚洲带绦虫囊尾蚴超微结构的影响[J].中国病原生物学杂志,2016,11(1):34-40.
[11]钟树怀,方文.猪囊尾蚴膜联蛋白基因的原核表达和免疫学分析[J].中国病原生物学杂志,2015,10(1):54-6,74.
[12]王丹,杨桂连,李显鹏,等.猪囊尾蚴重组诊断抗原的研究进展[J].吉林畜牧兽医,2010,31(5):16-9.
[13]王雪梅,骆江坤,李倩,等.猪囊尾蚴特异性抗原cC1重组耻垢分枝杆菌疫苗的构建与鉴定[J].中国血吸虫病防治杂志,2014,26(3):287-91.
[14]李婷婷,刘阳,苏永超,等.猪囊尾蚴疫苗的研究进展[J].山东畜牧兽医,2015,36(9):76-8.
[15]Hancock K,Khan A,Williams FB,et al.Characterization of the8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis[J].J Clin Microbiol,2003,41(6):2577-86.
[16]Scheel C M,Khan A,Hancock K,et al.Serodiagnosis of neurocysticercosis using synthetic 8-kD proteins:comparison of assay formats[J].Am J Trop Med Hyg,2005,73(4):771-6.
[17]Hubert K,Andriantsimahavandy A,Michault A,et al.Serological diagnosis of human cysticercosis by use of recombinant antigens fromTaenia soliumcysticerci[J].Clin Diagn Lab Immunol,1999,6(4):479-82.
[18]Chung JY,Bahk YY,Huh S,et al.A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis[J].J Infect Dis,1999,180(4):1307-15.
[19]Ferrer E,Bonay P,Foster-Cuevas M,et al.Molecular cloning and characterisation of Ts8B1,Ts8B2and Ts8B3,three new members of the Taenia solium metacestode 8kDa diagnostic antigen family[J].Mol Biochem Parasitol,2007,152(1):90-100.
[2]Espindola NM,Vaz AJ,Pardini AX,et al.Excretory/secretory antigens(ES)from in vitro cultures of Taenia crassiceps cysticerci,and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis[J].Ann Trop Med Parasitol,2002,96(4):361-8.
[3]王中全,崔晶.旋毛虫排泄分泌抗原的研究进展[J].中国预防兽医学报,2003,25(2):155-8.
[4]Ev LV,Maia AA,Pianetti G,et al.Immunological evaluation of a 26-kDa antigen fromTaenia soliumlarvae for specific immunodiagnosis of human neurocysticercosis[J].Parasitol Res,1999,85(2):98-102.
[5]Ito A,Plancarte A,Ma L,et al.Novel antigens for neurocysticercosis:simple method for preparation and evaluation for serodiagnosis[J].Am J Trop Med Hyg,1998,59(2):291-4.
[6]Yang HJ,Chung JY,Yun DH,et al.Immunoblot analysis of a10kDa antigen in cyst fluid of Taenia solium metacestodes[J].Parasite immunol,1998,20(10):483-8.
[7]Ko RC,Ng TF.Evaluation of excretory/secretory products of larval Taenia soliumas diagnostic antigens for porcine and human cysticercosis[J].J Helminthol,1998,72(02):147-54.
[8]Molinari JL,Garcia-Mendoza E,de la Garza Y,et al.Discrimination between active and inactive neurocysticercosis by metacestode excretory/secretory antigens of Taenia soliumin an enzymelinked immunosorbent assay[J].Am J Trop Med Hyg,2002,66(6):777-81.
[9]Del Brutto OH,Wadia NH,Dumas M,et al.Proposal of diagnostic criteria for human cysticercosis and neurocysticercosis[J].J Neurol Sci,1996,142(1):1-6.
[10]杜娟,牟荣,包怀恩,等.雷丸、阿苯达唑和吡喹酮对亚洲带绦虫囊尾蚴超微结构的影响[J].中国病原生物学杂志,2016,11(1):34-40.
[11]钟树怀,方文.猪囊尾蚴膜联蛋白基因的原核表达和免疫学分析[J].中国病原生物学杂志,2015,10(1):54-6,74.
[12]王丹,杨桂连,李显鹏,等.猪囊尾蚴重组诊断抗原的研究进展[J].吉林畜牧兽医,2010,31(5):16-9.
[13]王雪梅,骆江坤,李倩,等.猪囊尾蚴特异性抗原cC1重组耻垢分枝杆菌疫苗的构建与鉴定[J].中国血吸虫病防治杂志,2014,26(3):287-91.
[14]李婷婷,刘阳,苏永超,等.猪囊尾蚴疫苗的研究进展[J].山东畜牧兽医,2015,36(9):76-8.
[15]Hancock K,Khan A,Williams FB,et al.Characterization of the8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis[J].J Clin Microbiol,2003,41(6):2577-86.
[16]Scheel C M,Khan A,Hancock K,et al.Serodiagnosis of neurocysticercosis using synthetic 8-kD proteins:comparison of assay formats[J].Am J Trop Med Hyg,2005,73(4):771-6.
[17]Hubert K,Andriantsimahavandy A,Michault A,et al.Serological diagnosis of human cysticercosis by use of recombinant antigens fromTaenia soliumcysticerci[J].Clin Diagn Lab Immunol,1999,6(4):479-82.
[18]Chung JY,Bahk YY,Huh S,et al.A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis[J].J Infect Dis,1999,180(4):1307-15.
[19]Ferrer E,Bonay P,Foster-Cuevas M,et al.Molecular cloning and characterisation of Ts8B1,Ts8B2and Ts8B3,three new members of the Taenia solium metacestode 8kDa diagnostic antigen family[J].Mol Biochem Parasitol,2007,152(1):90-100.